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Master's Theses Theses and Dissertations

1969

The Etiology of Periodontal DiseaseRaymond Joseph LoiselleLoyola University Chicago

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Recommended CitationLoiselle, Raymond Joseph, "The Etiology of Periodontal Disease" (1969). Master's Theses. Paper 2373.http://ecommons.luc.edu/luc_theses/2373

THE ETIOLOGY OF PERIODONTAL DISF.ASE

by

Raymond Joseph Loiselle

A thesis submitted in partial fulfillment of the requirements

for the degree of Master of Science in Oral Biology.

Library -· Loyola University Medical Center June 8, 1969

ACIOOWLEDGMENTS

I wish to thank the faculty of Loyola University School of Dentistry for

their aid, encouragement and direction in this endeavor. Specific

individuals are numerous but acknowledgment of the many hours spent aiding

me are due to:

Dr. G. W. Rapp, Chairman of the Department of Oral Biology

Dr. P. D. Toto, Ch.airman of the Department of Oral Pathology

Dr. R. Locke, Department of Oral Biology

A number of individuals at the Veterans Administration Hospital, Hines,

Illinois, also aided me; and I would be remiss not to mention them:

Dr. R. J. Latronica, Oral Surgery Section

Dr. c. H. Stuever, Jr., Periodontics Section

Dr. L. Mayron, Dental Research Section

Dr. A. F. Goldberg, Oral Surgery Section

Dr. F. Frigan, Microbiology Section, Laboratory Service

ii

THE ETIOLOGY OF I'ER.IODONTAL DISEASE

TABLE OF CONTENTS

Introduction •

Specific Aims •

Experimental Method

Data • ., . Summary and Conclusion •

References •

•

•

• •

•

iii

1

7

9

. 13

. 19

• 20

INTRODUCTION

Periodontal disease consists of the destruction of the periodontal

tissues. These tissues include the gingiva, the alveolar bone, the

supporting bone, the periodontal ligament and part of the tooth. The

inflammation of the gingiva and underlying connective tissue spreads

apically to involve the alveolar bone and. process. These are attacked

resulting in resorption of the bone. The result of the reactions

ultimately causes the loosening and migration of teeth. Eventually, it

can result in loss of teeth.

Statistical studies performed in this country between the years

1960 to 1962 revealed the following data: Of this country's approximately

110,000,000 adults, more than 20,000,000 no longer had any teeth.

Approximately 25% of the remaining 90,000,000 had marked periodontal

disease. Fifty percent of the 90,000,000 had gingivitis. Only about 25%

of the adults exhibited little or no signs of gingivitis or periodontitis.

In the United States, only about lcrt.. of men or women aged 18·24 have a

severe form of periodontal disease. With aging, the disease develops

more rapidly. At 45-54 years of age 37% of the men and 30"L of the women

exhibited periodontitis while at 65-74 years, 58% of men and 33% of

women showed signs of periodontitis.

Studies in England revealed the steady increase in the incidence

of gingivitis from early childhood to late adolescence. The incidence of

periodontal lesions increases from 10-29% at 19~25 years of age up to

97-1007.. in persons aged over 45 years. Although severe manifestations

1

may not appear until middle age, the early lesion is frequently present

during childhood. Statistical studies indicate that periodontal disease

is the major cause of tooth loss. Al~hough caries is responsible for most

tooth removal prior to age 35, after this age over two-thirds of tooth

loss is due to periodontal disease.

At the 1966 World Workshop in Periodontics, it was stated that

practically all human beings have some form of periodontal disease.

Periodontal disease, per se, does not usually cause acute discomfort until

it enters into the terminal stages. Its public health significance lies

in its ultimate consequences, tooth loss. Thus, it becomes mandatory to

investigate the causes of the disease and the contributing factors to its

severity. It has been generally assumed that microorganisms or their

soluble components and/or products play a role in the etiology of perio­

dontal disease. Some of the evidence for and against this are as follows:

Several authors have indicated that some of the bacteria in the

oral flora produce enzymes that are capable of disrupting gingival tissue

components (Schultz-Haudt and Lundquist 1962, Lucas, R. B. and Thonard; J.C.

1955, Schultz-Haudt, S. D. and Scherp, H. W. 1955 A, Schultz-Daudt, S. D.

and Scherp, H. W. 1955 B, Schultz-Haudt, S. D. and Scherp, H. W. 1956,

Schultz-Haudt, S. D., Bibby, B. G. and Bruce, M. A. 1954, Rapp, G. W.

and Orzolek, L. M. 1966). However, several investigators have since

pointed out that these same destructive enzymes are also produced by

p0lynX>rphonuclear leucocytes, which have been found in inflamed tissue.

(Lazarus, G. s., Brown, R. s., Daniels, J. R. and Fullmer, H. M. 1968).

2

S. W. Leung (1962) has made the statement that "the initial injury

which starts the chain of events is thought to be physical damage caused by

such substances as calculus and impacted food and debris. This leads to

inflammation and subsequent bacterial invasion of the injured tissues."

However, dental calculus deposition has been shown to take place in germ­

free rats. Baer and Newton demonstrated in both Swiss germ-free and

conventional mice (1960) there was downgrowth of the epithelial attachment

and loss of alveolar bone. Inflammation and the presence or absence of

bacteria did not seem to play a role, initially, in these periodontal

changes.

Keyes and Jordan (1964) have found filamentous organisms capable of

producing an active type of periodontal disease in albino hamsters. These

filamentous organisms have been recovered from subgingival plaque of

susceptible strains of hamsters.

There have been conf lieting reports concerning the penetration of

bacterial into the gingival tissue. Gibson and Shannon (1965) demonstrated

that if tooth debridement was performed in the presence of carbon particles,

the particles appeared to penetrate the underlying connective tissues as

seen in stained tissue sections. This effect did not appear in the biopsies

and sections from the non-debrided group. This may tend to refute some

histological studies which purported to demonstrate bacteria in gingival

tissues (Beckwith et al 1925, Beckwith et al 1927, Box 1950, Vargas, B.

et al 1959). It has also been reported that microorganisms have been

forced into the tissues by the sectioning procedures, thus creating the

artifact of bacterial presence in the tissues in the finished histological

3

slide.

Diffusable bacterial products are also agents in the etiology of

periodontal disease. (Bibby, BG 1960). One example of this is the

demonstration by Cobb and Brown that a sterile, filterable substance from

gingival sulci and periodontal pockets is toxic to cells in tissue culture.

A greater effect was observed with the material from the periodontal pockets

(Periodontics 5:5, 1967). Rizzo (1967) demonstrated that hydrogen sulfide

is produced in periodontal pockets.

Studies have shown the presence of fungal organisms in addition to

bacteria in calculus and plaque. Howell, Rizzo, and Paul (1965) cultured

calculus-plaque material and found bacteria and the fungal organisms

(Actingmyces israelii, Actinpm,yces naeslundii and Veillonella species

(schizomycetes), Wenford and Haberman (1966) cultured diseased gingiva and

isolated strains of ActiD.O!!Y'ces naeslundii and Nocardia salivae, as well

as bacterial microorganisms. However, the presence of these fungal organisms

is no indication of their role, if any, in the etiology of periodontal

disease.

Severe periodontal disease was induced in a group of mice infected at

birth with polyoma virus (1965). Shklar and Cohen described the periodontal

alterations, which included apical migration of epithelial attachment,

periodontal pocket formation with purulent exudate, and extensive resorption

of alveolar bone. Similarly, Baer and Kilham (1964) describe the occurrence

of periodontal disease in hamsters inoculated intracerebrally with rat

virus.

Rizzo and Mergenhagen (1965) produced hypersensitivity reactions in

4

a palato-gingival site in the rabbit by sensitizing and challenging with

horse serum, and by sensitizing with human tubercle bacilli and challenging

with PPD. The ensuing plasmacytosis, enhanced by repeated local injections

of antigen, resembled histologically that occurring in human gingivitis.

In a later study Rizzo and Mitchell (1966) reported that the repeated

deposition of antigenic protein into healthy gingival pockets of the

rabbit caused "chronic allergic inflanmation", with plasmacytosis, in the

adjacent periodontal tissues. Low levels of serum antibody to the deposited

antigen were evident within two weeks after treatment began and tended to

remain at low levels throughout the 17 weeks of continual pocket exposure

to the antigen, egg albumin. The published data indicate that antigen was

absorbed into the gingiva and that it caused both a local and systemic

immunologic response. Thonard and Dalbow (1965) injected sheep red blood

cells into the interdental papillae of rats and guinea pigs and found

antibody-forming cells in the gingival tissue by preparing cell suspensions

and testing for antibody production by the antibody-plaque technique.

Dalbow and :Baumhammers (1968) showed by immunofluorescent techniques

the presence of major classes of immunoglobulins in periodontal tissue of

both humans and animals with periodontal disease. These immunoglobulins

were also present in normal human gingiva.

Platt, Dalbow and Crosby (1968) upon trying to simulate a delayed­

hypersensitivity response by the injection of histo-compatible lymphoid

cells into germ-free mice intraperitoneally, followed by the intragingival

injection of heat-denatured collagenase in Freund's adjuvant, were able

to d8Dl>nstrate marked round-cell infiltration, pronounced collagen

5

disaggregation and fibrotic changes in the gingivae.

Ranney (1968) challenged sensitized monkeys by inserting ovalbumin

into the gingival sulcus. Three hours after a single challenge, a dense

infiltration of polymorphonuclear leucocytes beneath the sulcular epithelium

and vascular congestion and dilatation were visible. In three days, the

acute inflammation subsided and mononuclear cells were prominent in the

tissue sections. When three consecutive challenges were employed the

cellular infiltration was more dense and dissolution of collagen and

osteoclastic activity were prominent.

Thonard, Welty, Dalbow and Platt (1968) gave genn-free mice a pro­

longed regimen of challenge with Clostridiue histolyticum collagenase, in

the native state or heat-inactivated, and showed an altered tissue response

that histo•pathologically resembles chronic periodontitis. If inmunosuppres­

sive quantities of Imuran were given to the animals, no significant changes

in the periodontal structures occurred, even when subjected to prolonged

challenge with the enzyme.

Thus. immunological reactions have been shown to cause inflammation

and tissue alterations in experimental animals. The nature of these

reactions has not been identified, nor have their relations, if any, to

human periodontal disease. Although proper oral hygiene will alleviate some

of the symptomatology of periodontitis, the relationship of bacteria and

fungi to the disease have not been satisfactorily explained.

6

SPECIFIC AIMS

The presence of immunoglobulins in the crevicular fluid of human

gingiva as reported by Brill and Bronnestam (1960) and confirmed by other

investigators coupled with the occurrence of immunoglobulins in the cyto­

plasm of gingival epithelial cells raised the following questions: (1)

What is the nature of the antigen or antigens responsible for stimulating

the local antibody production? (2) Is this local production of

immunoglobulin associated with a protective or an allcrrgic reaction on the

behalf of the host? It may be that the globulins are directed against

bacterial antigens or against tissue components. When viewed in this

context, the problem of cross-reactivity of antibodies to bacterial and

tissue antigens may play a major role in chronic periodontal disease. In

other words, antibodies initially formed against bacteria may ultimately

cross react tissue in the periodontal structure. The patients utilized in

this clinic study were selected from the population examined by the Dental

Service, Veterans Administration Hospital, Hines, Illinois. The patients

utilized were those showing no acute cause for periodontitis. The lack of

the acute phase of the disease at the time of sampling did not imply it did

not exist, but merely that it had been treated and eliminated and what was

now seen was a chronic disease state after the removal of acute initiating

factors. The diagnosis of chronic periodontitis was confirmed by histolog­

ical studies of biopsy specimens taken after cultures of the gingival crevicj

were obtained.

This particular study is designed to examine one aspect of periodontal

7

disease - chronic, non-supperative periodontitis. I wish to determine

whether there is a relationship between the bacterial organisms present in

the gingival sulcus of patients with chronic, non-supperative periodontitis

and the reaction which these patients have to periodontal therapy.

8

EXPERIMENTAL METHOD

Gingival culture specimens were taken in a standardized fashion from

twenty patients. The technique used utilized pre-packaged individual

sterile trays for each patient. The cultures were taken by carefully

inserting sterile absorbant paper points into the gingival crevice with

sterile cotton forceps. The paper points were then placed in test tubes

containing enriched nutrient broth and transported in this media to the

bacteriology laboratory. In the bacteriology laboratory the points were

removed from the test tube and plates streaked and incubated for the purpose

of detecting all anaerobic and aerobic organisms present in the initial

specimen taken from the gingival crevice. In addition, thioglycolate broth

was also inoculated as a further means of checking the effectiveness of

culturing the organisms in the aerobic and anaerobic techniques. Using

selective media and environment all organisms found growing on any of the

plates were identified.

The organisms routinely cultured from all of the patients sampled

were streptococcus v!ridans. The Streptococcus viridans were then further

subcultured and identified. Identification of Streptococcus mitis and

Streptococcus salivarius was made by the use of selective Streptococcus

mitissalivarius medium. Neissena catarrhalis were recovered from 30% of the

patients.

The distinction between s. salivarius and s. mitis was made on the

basis of morphology, growth in selective media and ability to ferment and

utilize added nutrients. S. salivarius are spherical or ovoid cells less

than 1 micron in diameter and form either long or short chains. Normally

9

an indifferent or gamma hemolytic reaction on horse blood agar is seen but

a few strains wi 11 produce greening. Optimal growth occurs at 370 and no

growth occurs at 100 or 470 C. When grown on agar media containing sucrose

or raffinose, large characteristic mucoid colonies are formed. These

colonies may be rough or smooth. Variation between rough and smooth types

occurs spontaneously in broth cultures. S. salivarius will not grow in

broth containing 6.5% sodiUl'll chloride or at a pH 9.6 or on 3<11. bile blood

agar or in milk containing 0.1% methylene blue. S. Salivarius ferments

salicint inulin, raffinose, sucrose, maltose and glucose. Gelatin in

starch is not hydroli&ed by s. salivarius nor can it produce ammonia from

arginine.

Streptococcus mi.tis forms slightly smaller cells than s. salivarius,

seldom exceeding 0.8 microns in diam.eter. Only a fw strains of S. mi.tis

produce mucoid colonies on sucrose agar. True rough And smooth variants

occur regularly in solid or liquid media and the rough form tends to revert

to the smooth form in liquid media. s. mitis consistently ferments glucose,

maltose and sucrose, usually ferments lactose and salicin, and rarely

ferments ra:ffinose. Gelatin. is not hydrolized by S. mitis.

'11le large number of organisms necessary for further stages of this

work were obtained by propagating pure cultures of S. 84livarius ATCC9222,

S. mitis ATCC903 and N. catarrhalis ATC8176 on selective media plates.

The colonies were then harvested mechanically be means of sterile loop from

the plate into sterile BHI broth where they were re-suspected. The

organisms were then recovered from the broth by centrifugation. Specific

antibodies to the bacteria were then developed in New Zealand rabbits.

10

Additional quantities of the bacteria were grown for conjugation with

fluorescein isothiocynate.

Gingival biopsies and biopsies of masticatory mucosa from edentulous

regions were performed in the patients from whom the cultures were taken.

Frozen sections of these tissues were cut for microscopic and immunologic

examination.

It was of interest to know if there were streptococcal antigens or

antibodies to these antigens in the tissue sections. The sections were

cut, frozen and mounted on a glass micrvscope slide. The tissue was

treated with rabbit antiserum to the specific microorganisms (nd.tis and

salivar!us) and then stained with fluorescein conjugated goat or sheep

anti-rabbit serum.

To reduce the non-specific binding of the rabbit anti•mitis and

anti-salivarus serum to the tissue, each step was preceded by incubation

with normal serum. Fluorescein tagged mieroorganisms were also used to

deterllline the presence of antibodies to s. mitis and s. salivarius in the

tissue sections.

The results obtained in the two variations above with human gingival

tissue was compared with that seen in masticatory mucosa from the edentu-

lous tuberosity from the same patients. Thus, the tissue from theedentulous

region should not demonstrate the same immunological reactions as the

gingival tissue, not having been subjected to the same microbiological and

trauma stresses as the gingival tissue. The alternative to using human

tissue as a control would be to use the gingival tissue of an immunized and

a nonimmunized, non-exposed rabbit. This would assure both positive and

negative controls, especially for the determination of antibodies in the 1

tissue but would not necessarily be of value in relation to human periodon­

tal disease.

The immunological procedures used for staining tissue are as

follows:

(1) tissue ~rabbit antiserum ~ normal sheep serum ~ fluorescent

sheep antiserum to rabbit;

(2) tissue ..::.. fluorescent microorganisms.

12

DATA

A coupling of the specific anti-sera was noted in the gingival

sections while none was present in the tissue specimens of masticatory

mucosa from edentulous regions. Figures 1 through 4 show fluorescent

staining with anti-neisseria and anti-streptococcus sera. Figure 5 shows

binding of fluorescein tagged bacteria (S. mitis) to the tissue section.

Histologic examination of comparable sections stained with H & E show a

severe plasmacytosis. The same patients masticatory mucosa tissue sections

did not show a marked plasmacytosis nor did they bind the specific anti-sera

or fluorescein coupled Streptococcus mitis.

13

Figure 1

Gingival tissue from a patient with periodontal disease stained with rabbit antiserum to Neisseria cat arrhalis , followed by fluorescein-conjuga ted sheep, anti-rabbit-globulin serum. This shows fluorescence in cella belo:w the keratin layer. Magnification about lOOX.

14

Figure 2

Gingival tissue from a patient with periodontal disease stained with rabbit antiset'U!ll to Neisseria catarrbalis, follwed by fluorescein-conjugated sheep, anti-rabbit-globulin serum. This show fluorescence in cells below the keratin layer. Magnification about l OOX.

15

Figure 3

Gingival t issue from a patient with periodontal disease stained with rabbit antiserum to Streptococcus mitis , followed by fluorescein con jugated sheep anti .. rabbit-globulin serum. This shows fluorescence exhibited by a single cell in the field. Magnification = 250X.

16

Figure 4

Gingival tissue from a patient with periodontal disease stained with rabbit antiserum to Streptococcus salivarius , followed by fluorescein-conjugated sheep anti·rabbit-globulin serum. This fluorescence is seen in the sub~ keratin layer . Magnif i cation = 400X.

17

Figure 5

Gingival tissue from a patient with periodontal diseas.e stained with fluorescefn .. conjugated Streptococcus mi.tis . Fl uorescence is seen throughout the whole epi t hel i al layer . Magnification ~ 25 .

18

SUMMARY AND CONCLUSIONS

1) Organisms cultured from the gingival crevice of patients with non­

superative, chronic periodontitis were predominently Streptococcus viridans.

Neisseria catarrhalis were recovered from 307o of the patients.

2) Further identification of the S. viridans showed them to be S. mi tis and

s. salivarius.

3) Anti-sera to s. mitis and s. salivarius and the bacteria themselves

could be bound to sections of mucosa from the gingiva of the patients but

not to the sections of masticatory mucosa from ed.entulous regions of the

same patients.

From these points I concluded that the bacteria in the gingival sulcus

plays a role in chronic periodontal disease. This role would appear to be

in the realm of immunologic reactions rather than a toaic reaction.

19

Baer, P. N. and Kilham, L. Aged Hamster. Oral Surg.

Rat Virus and Periodontal Disease. 18:803-811 {Dec) 1964.

IV. The

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Baer, P. N. and Newton, w. L. Studies on Periodontal Disease in the Mouse. III. The Germ-free Mouse and its Conventional Control. Oral Surg. 13:1134-1144 (Sept) 1960.

Beckwith, T. D., Simonton, F. V. and Williams, A. A Histologic Study of the Gums in Pyorrhea. J. Amer. Dent. Assoc. 12:129-153 (Feb) 1925.

Beckwith, T. D. > Simonton, G. W. and Rose, E. J. The Presence of Bacterial Microorganisms in Human Gingival Tissue in Gingivitis. Dent. Cosmos 69:164-171 (Feb) 1927.

Bibby, B. G. Role of Bacteria in Periodontal Disease. J. Tenn. Dent. Assoc. 40:287-295 (Oct) 1960.

Box, H. K. Gingtval Clefts and Associated Tracts. New York State Dent. J. 16:3-10 (Jan) 1950.

Brill, N. and Bronnestam, R. Immuno•electrophoretic Study of Tissue Fluid from Gingival Pockets. Acta Odont. Scandinavica 18:95-100 {Aug) 1960.

Cobb, c. M. and Brown, L. R. The Effect of Exudate from the Periodontal Pocket on Cell Culture. Periodontics 5:5·18 (Jan-Feb) 1967.

Courant, P. R. and Bader. H. Bacteroides Melaninogenicus and Its Products in the Gingiva of Man. Periodontics 4:131•136 (May-June) 1966.

Dalbow, M. H. and Bauhammers. A. Immunocytology of Periodontal Disease. IADR Abstract #405 (.t-1.arch) 1968.

Gibson, W. A. an.d Shannon, I. L. Simulation with Carbon Particles of Bacterial Infiltration of Human Gingival Tisaues. Periodontics 3:57-9 (Mar-April) 1965.

Howell. A. Jr., Rizzo, A. A. and Paul, F. Cultivable Bacteria in Developing and Mature Human Dental Calculus. Arch. Oral Biol. 10:307-13 (Mar-April) 1965.

Jordan, H. V. and Keyes, P. H. Periodontal Lesions in the Syrian Hamster. III. Findings Related to an Infections and Transmissible Component. Arch. Oral Biol. 9:372-400 (July-Aug) 1964.

20

Jordan, H. V. and Keyes, P. H. Aerobic, Gram-positive, Filamentous Bacteria as Etiologic Agents of Experimental Periodontal Disease in Hamsters. Arch. Oral Biol. 9:401-414 (July-Aug) 1964.

Lazarus, G. s., Brown, R. S., Daniels, J. R. and Fullmer, H. M. Human Granulocyte Collageuaae. Science 159:1483-4 (March 29) 1968.

Leung, s. W. Relation of Calculus, Plaque and Food Impaction to Periodontal Disease. J. Dent. Res. 41:306-319 (Jan-Feb) 1962.

Lucas, R. B. and Thon.ard, J. C. The Action of Oral Bacteria on Collagen. J. Dent. Res. 34:118-122 (Feb) 1955.

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Ranney, R. R. Periodontal Lesions in Sensitized Squirrel Monkeys Following Challenge Through the Gingival Sulcus. IADR Abstract 1407 (March) 1968.

Rapp, G. W. and Orzolek, L .. M. Protease Activities of Normal and Inflammed Human Gingiva. J. Perio. 37:331-4 (Jul-Aug) 1966.

Rizzo, A. A. Research on Periodontal Disease 1963-1965. J. A. D. A. 72:1427-38 (June) 1966.

Rizzo, A. A. The Possible Role of Hydrogen Sulfide Production in Periodontal Pockets. Periodontics 5:233-6 (Sept-Oct) 1967.

Rizzo, A. A. and Mergenhagen, s. E. Histopathologic Effects of Endotoxin Injected. into Rabbit Oral Mucosa. A:rch. Oral Biol. 9:659-70 (Nov-Dec) 1964.

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Rizzo, A. A. and Mitchell, C. T. Chronic Allergic Inflammation Induced by Repeated Deposition of Antigen in Rabbit Gingival Pockets. Periodontics 4:5-10 (Jan-Feb) 1966.

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21

Schultz-Haudt and Scherp, H. W. Lysis of Collagen by Human Gingival Bacteria. Proc. Soc. Exp. Biol. and Med. 89:697-700, 1955. B

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Shklar, G. and Cohen, M. M. The Development of Periodontal Disease in Experimental Animals Infected with Polyoma Virus. Periodontics 3:281-285 (Nov-Dec) 1965 ..

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World Workshop in Periodontics, Ann Arbor Michigan June 6-9, 1966.

22

I I

APPROVAL SHEET

The thesis submitted by Dr. Raymond Joseph Loiselle has been

read and approved by members of the Department of Oral Biology.

The final copies have been examined by the director of the

thesis and the signature which appears below verifies the fact that

any necessary changes have been incorporated, and that the thesis is

now given final approval with reference to content, form, and

mechanical accuracy.

The thesis is therefore accepted in partial fulfillment of the

requirements for the Degree of Ylaster of Science.

Date Gustave W. Rapp, -.Ph. D. Cho.irman, Department of

Oral Biology